Compositions for treating biofilm

ABSTRACT

A two component composition comprises an anchor enzyme complex to degrade biofilm structures and a second anchor enzyme component having the capability to act directly upon the bacteria for a bactericidal effect.

CROSS REFERENCE TO THE RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/587,818 filed Jun. 6, 2000, which is a continuation-in-part of U.S.application Ser. No. 09/249,674 filed Feb. 12, 1999, which is acontinuation-in-part of U.S. application Ser. No. 08/951,393 filed Oct.16, 1997 (issued as U.S. Pat. No. 5,871,714 on Feb. 16, 1999), all ofwhich are incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

Standard chemical analyses, traditional microscopic methods as well asdigital imaging techniques such as confocal scanning laser microscopy,have transformed the structural and functional understanding ofbiofilms. Investigators, with these techniques have a clearerunderstanding of biofilm-associated microorganism cell morphology andcellular functions. The heightened awareness of metabolic biochemistryand the events associated with them have led to a better understanding,not only of individual cells and their varying environments, but alsocollections of cells that form colonies. Further, certain relationshipsof colonies to each other are under the direct influence of the biofilmin which they reside.

Concurrent with the increased understanding of cellular activity andinter-colony relationships, there has been an awareness developed aboutthe biofilm in which the cells reside. While there has been an increasedunderstanding of the architecture and composition of the biofilm matrix,the most significant advances have occurred in the inter-relationshipsamong cells, colonies and biofilm matrices. Indeed, the basis of oneaspect of this invention is founded in the integration of theenlightened understanding of microorganism activity within the influenceof the biofilm in which they reside.

Biofilms are matrix-enclosed accumulations of microorganisms such asbacteria (with their associated bacteriophages), fungi, protozoa andviruses that may be associated with these elements. While biofilms arerarely composed of a single cell type, there are common circumstanceswhere a particular cellular type predominates. The non-cellularcomponents are diverse and may include carbohydrates, both simple andcomplex, proteins, including polypeptides, lipids and lipid complexes ofsugars and proteins (lipopolysaccharides and lipoproteins).

For the most part, the unifying theme of non-cellular components ofbiofilms is its backbone. In virtually all known biofilms, the backbonestructure is carbohydrate or polysaccharide-based. The polysaccharidebackbone of biofilms serves as the primary structural component to whichcells and debris attach. As the biofilm grows, expands and ages alongbiologic and non-biologic surfaces in well-orchestrated enzymaticsynthetic steps, cells (planktonic) and non-cellular materials attachand become incorporated into the biofilm. The growing biofilm not onlyattracts living cells; it also captures debris, food particles, cellfragments, insoluble macromolecules and other materials that add to thelayer upon the polysaccharide backbone. In this fashion, layeringcontinues and is repeated so that the initial layers i.e., those closestto the original polysaccharide backbone, become buried or embedded inthe biofilm. As the biofilm ages, there are layers upon layers ofpolysaccharide backbone with the attendant cells, debris and insolublemacromolecular structures.

Biofilms are the most important primitive structure in nature. In amedical sense, biofilms are important because the majority of infectionsthat occur in animals are biofilm-based. Infections from planktonicbacteria, for example, are only a minor cause of infectious disease. Inindustrial settings, biofilms inhibit flow-through of fluids in pipes,clog water and other fluid systems and serve as reservoirs forpathogenic bacteria and fungi. Industrial biofilms are an importantcause of economic inefficiency in industrial processing systems.

Biofilms are prophetic indicators of life-sustaining systems in higherlife forms. The nutrient-rich, highly hydrated biofilms are not justlayers of plankontic cells on a surface; rather, the cells that are partof a biofilm are a highly integrated “community” made up of colonies.The colonies, and the cells within them, express exchange of geneticmaterial, distribute labor and have various levels of metabolic activitythat benefits the biofilm as a whole.

Planktonic bacteria, which are metabolically active, are adsorbed onto asurface which has copious amounts of nutrients available for the initialcolonization process. Once adsorbed onto a surface, the initialcolonizing cells undergo phenotypic changes that alter many of theirfunctional activities and metabolic paths. For example, at the time ofadhesion, Pseudomonas aeruginosa (P. aeruginosa) shows upregulated algC,algD, algU etc. genes which control the production of phosphomanomutaseand other pathway enzymes that are involved in alginate synthesis whichis the exopolysaccharide that serves as the polysaccharide backbone forP. aeruginosa's biofilm. As a consequence of this phenotypictransformation, as many as 30 percent of the intracellular proteins aredifferent between planktonic and sessile cells of the same species.

In summary, planktonic cells adsorb onto a surface, experiencephenotypic transformations and form colonies. Once the colonizing cellsbecome established, they secrete exopolysaccharides that serves as thebackbone for the growing biofilm. While the core or backbone of thebiofilm is derived from the cells themselves, other components e.g.,lipids, proteins etc, over time, become part of the biofilm. Thus abiofilm is heterogeneous in its total composition, homogenous withrespect to its backbone and heterogeneous with respect it its depth,creating diffusion gradients for materials and molecules that attempt topenetrate the biofilm structure.

Biofilm-associated or sessile cells predominate over their planktoniccounterparts. Not only are sessile cells physiologically different fromplanktonic members of the same species, there is phenotypic variationwithin the sessile subsets or colonies. This variation is related to thedistance a particular member is from the surface onto which the biofilmis attached. The more deeply a cell is embedded within a biofilm i.e.,the closer a cell is to the solid surface to which the biofilm isattached or the more shielded or protected a cell is by the bulk of thebiofilm matrix, the more metabolically inactive the cells are. Theconsequences of this variation and gradient create a true collection ofcommunities where there is a distribution of labor, creating anefficient system with diverse functional traits.

Biofilm structures cause the reduced response of bacteria to antibioticsand the bactericidal consequences of antimicrobial and sanitizingagents. Antibiotic resistance and persistent infections that arerefractory to treatments are a major problem in bacteriologicaltransmissions, resistance to eradication and ultimately pathogenesis.While the consequences of bacterial resistance and bacterialrecalcitrance are the same, there are two different mechanisms thatexplain the two processes.

The use of enzymes in degrading biofilms is not new. Compositionalpatents as well as published scientific literature support the conceptof using enzymes to degrade, remove and destroy biofilms. However, thelack of consistency in results and the inability to retain the enzymesat the site where their action is required has prohibited theirwidespread use.

As an alternative to enzymes, harsh chemicals, elevated temperatures andvigorous abrasion procedures are preferentially used over enzymes. Thereare conditions, however, where these non-enzymatic approaches cannot beused e.g., caustic- and acidic-sensitive environments, temperature orabrasion sensitive components that are associated with the biofilm anddynamic fluid action. When a biofilm is growing in an area where thereis a constant fluid flow, the agents that remove biofilms are flushedaway before they can carry our their desired function. This isparticularly true for medical situations where aggressive sterilizationprocedures cannot be carried out and there is a desired fluid flow.

Removing and controlling biofilm growth in biologic media arespecifically sensitive to harsh treatments. Biofilms in the oral cavity,on implanted devices and infections that occur in the alimentary andvaginal tracts or in eyes, ears etc. are particularly suited for anenzymatic treatment. There are also specific disease conditions, such aspneumonia and cystic fibrosis which are bacteria-based and occur in thelung, that would benefit from an enzymatic treatment only if the enzymescould be retained at the site long enough to fully realize theirtherapeutic actions.

Biofilm growth and the proliferation of infections in biologic systemsare particularly sensitive to fluid-flow dynamics. Specific organs whereinfections occur e.g. eyes, oral cavity, gastrointestinal tract, vaginaltract, lungs etc., fluid and mucus flow is an integral part of thesystem's normally functioning mode. Consequently, it is desirable tohave the capability of removing unwanted biofilms in a non-harsh way inwhich the agent that acts on the biofilm is retained in close proximityto the biofilm and not swept away by fluids that are integral to thefunctioning system.

There are situations in or related to biologic systems where flow isminimal or non-existent. In these circumstances, the lack ofdemonstrated efficacy of enzymes to control biofilms is not relatedexclusively to their lack of ability to be retained at the site of thebiofilm. Rather, the choice of enzyme to degrade the biofilm wasinappropriate. An example is biofilm control on contact lenses and thecases or containers that hold the lenses when they are not in use. Inthese circumstances, it may not be a mandatory requirement for a meansto retain the enzymes at or near the biofilm structure but only that theappropriate enzyme be part of the enclosed system.

It is also desirable to not only be able to degrade a biofilm within abiologic system, but also to be able to have a direct effect on thebacterial cells that are released as the biofilm is undergoingdegradation. The combination of biofilm degradation and agents thatdirectly affect bacterium is also not a new strategy. However, notinfrequently in an open system, the same forces that flush or sweep awaythe biofilm degrading enzymes also flush bactericidal agents so thatthey cannot act directly upon bacteria unless there is a chance meetingbetween the agent and a planktonic bacterium.

SUMMARY OF THE INVENTION

Antibiotic/Antimicrobial Resistance. In the case of antibiotic orantimicrobial resistance, biofilms provide the unique opportunity forbacteria to reside in close proximity with one another for long periodsof time. This prolonged juxtaposition of bacteria allows gene transferbetween and among bacteria, allowing the genes of resistance to betransferred to same or different strains of bacteria to neighboringcells that are not resistant. Consequently, a virulent cell can transferits virulence genes to a non-virulent cell, making it resistant toantibiotics.

Antibiotic/Antimicrobial Recalcitrance. In the case of antibiotic orantimicrobial recalcitrance, there are two possible explanations, bothof which involve the biofilm and both of which may be operativesimultaneously. While gene transfer may occur, it is not a factor inrecalcitrance.

The first of the explanatory mechanisms is simply a physical phenomenon:the biofilm structures present a barrier to penetration of antibioticsand antimicrobial agents and a protective shroud to physical agents suchas ultraviolet radiation. The biofilm, with its polysaccharide backboneand residual debris that is associated with the biofilm, provides abarrier to deep-seated bacteria. Unless the biofilm is removed ordisrupted, complete cellular kill within the biofilm structure is notachieved by chemical or physical agents.

The second explanatory mechanism is based on biochemical or metabolicprinciples. Just as the deep-seated bacteria are protected from chemicaland physical agents by the “barrier” effect of the biofilm, the biofilmalso acts as a barrier to nutrients that are necessary for normalmetabolic activity. Further, the nutrient-limited bacteria are in areduced state of metabolic activity, which make them less susceptible tochemical and physical agents because the maximal effects of thesekilling agents are achieved only when the bacteria are in ametabolically active state.

With any of the possible mechanistic explanations for either resistanceor recalcitrance, removal or disruption of the biofilm is a mandatoryrequirement. Stripping away of the biofilm components e.g., thepolysaccharide backbone with the accumulated debris accomplishes severalobjectives: 1) reduced opportunity for gene transfer; 2) increasedpenetration of chemical and physical agents; and 3) increased free-flowof nutrients which would elevate the metabolic activity of the cells andmake them more susceptible to chemical and physical agents. Furthermore,removal or disruption of the biofilm will free cells from a sessilestate to make them planktonic which also increases their susceptibilityto chemical and physical agents.

Prevention of Biofilm Formation. Under ideal conditions for controllingbiofilms, the preferred approach for limiting the detrimental effects ofbiofilms is prevention of initial colonization by cells. For the mostpart, these approaches focus on the environment in which planktonicbacteria are present without particular attention to the bacteriathemselves. This can be done to a limited extent through physical meanse.g., electrical charges etc., chemical strategies e.g., surfacecoatings (paints and varnishes with antimicrobial chemicals) etc. andbiochemical means e.g. nutrient limitation. However, for the majority ofsituations when fouling by biofilms occurs, these strategies are notpractical or at best have limited utility.

Limiting Early Biofilm Growth. The next line of defense against theadverse effects of biofilms revolves around curtailing the consequencesof the post-initial colonization of planktonic bacteria to a surface bylimiting the initial proliferation of the biofilm. This can beaccomplished, only to a limited extent, by continual disruption ofearly, immature biofilms or by inhibiting the biosynthesis of thestructural exopolysaccharide backbone. Interdiction of earlyexopolysaccharide synthesis is usually achieved by macrolide antibioticse.g., large ring lactones, erythromycin being one example. This latercourse of action constitutes a shift from an attempt to control thebiofilm structure or environment to a direct action upon the livingcells within the biofilm.

Destroying Established Biofilms. For established biofilms, with variouslevels of embedded cells, disruption, fragmentation and removal of thebiofilm is necessary. This can be accomplished, under limitedcircumstances, with physical means e.g., abrasion methods, sonication,electrical charge stimulation, detergent and enzymatic. There areobvious drawbacks to any one method, precluding a universal method orapproach. However, the common trait of all of these methods lies intheir focus on the biofilm structure and not the living cells within thebiofilm.

If, by any one of the methods, the structure of the biofilm is alteredor disturbed, a secondary, complementary attack on the living cellswithin the biofilm can be made with antibiotics and antimicrobialagents.

An important aspect of the invention lies in two concepts, both of whichmay operate independently, but when combined, they effectively removebiofilms and prevent their reestablishment. The first of these is theremoval of the biofilm structure in an orderly and controlled manner.The second concept is a specific consequence of removing the biofilmstructure. During the removal or dismantling of the biofilm structure,especially the exopolysaccharide backbone, cells within the biofilmbecome more susceptible to the bactericidal action of antimicrobials,antibiotics, sanitizing agents and host immune responses. As the biofilmis removed, some cells within the biofilm are liberated and becomeplanktonic; others, however, remain sessile but are more vulnerable tobeing killed because the protective quality of the biofilm is reduced.

One aspect of the invention consists of one or more hydrolytic enzyme(s)whose specificity includes its (their) ability to degradeexopolysaccharide backbone structure(s) of a biofilm produced bybacterial strain(s). Attached to the enzyme(s), either through chemicalsynthetic procedures or recombinant technology, are one or more moietiesthat have the capability of binding, either reversibly, in anon-covalently, or irreversibly (covalent bonded) to a surface near thebiofilm or the biofilm itself. This aspect is directed at thedegradation of the biofilm backbone structure.

Another aspect of the invention consists of two or more hydrolyticenzymes. One enzyme has the specificity to degrade the biofilm'sexopolysaccharide backbone structure of a biofilm; at least one otherenzyme is hydrolytic in nature, having the capability to degradeproteins, polypeptides, lipids, lipid complexes of sugars and proteins(lipopolysaccharides and lipoproteins). Attached to the enzymes, eitherindividually or collectively as a single unit through chemical syntheticprocedures or recombinant technology, are one or more moieties that havethe capability of binding either reversibly, non-covalently, orirreversibly (covalent bonded) to a surface near the biofilm or thebiofilm itself. This aspect is directed at the degradation and removalof the biofilm backbone structure along with any other materials thatmay be associated with the backbone, which collectively constitute theentire biofilm.

Still another aspect of the invention consists of two or more enzymes,wherein at least one enzyme has the capability of degrading a biofilmstructure produced by a bacterial strain, or a mixed combination ofvarious strains, and the other enzymes(s) has (have) the capability ofacting directly upon the bacteria, causing lysis of the bacterial cellwall. One or more moieties are attached to the enzymes, forming either asingle unit or multiple units. The moieties are attached to the enzymeseither through chemical synthetic procedures or recombinant technologyto give the enzyme moiety the capability of binding either reversibly,non-covalently, or irreversibly (covalent bonded) to a surface near thebiofilm or the biofilm itself. The purpose of this multi-enzyme systemis directed at the degradation and removal of the biofilm with thecontemporaneous bactericidal consequences for bacteria that wereembedded in the biofilm's structure.

A fourth aspect of the invention consists of two sets of enzymes, thefirst being one or more enzymes with the appropriate anchor attached tothe enzyme(s) for the purpose of degrading the biofilm structure; thesecond set of enzymes are also connected to anchor molecules whosefunction is to generate active oxygen to directly attack and killbacteria that are exposed during the process of the degradation andremoval of the biofilm.

A fifth aspect of the invention consists of one or more enzyme complexesto degrade biofilm structures and a second component of one or moreunbound or free non-enzymatic bactericidal components whose function isto kill newly exposed bacteria as the biofilm structure is removed. Thenon-enzymatic bactericidal agents include, but are not limited to,antimicrobial agents, antibiotics, sanitizing agents and host immuneresponse elements.

The purpose of these various embodiments is to hold or retain thebiofilm-degrading enzymes and bactericidal components in fluid-flowsystems that are open, partially open or, at least not completely closedsystems. Without the capability to keep the appropriate active agents ator near the biofilm structure, they may be swept away in the fluid flow.

The above five previously described aspects of the invention apply toopen or partially open systems where there is fluid flow. However, thereis also an additional embodiment for completely closed systems in whichthe enzyme or antibacterial agent may or may not have a binding moietyattached to.

A sixth aspect of the invention consists of one or more appropriatelyselected enzymes, not being connected to a binding moiety but limited bytheir ability to degrade a biofilm that is contained within such aclosed system where there is minimal to no fluid flow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a biofilm from a distance.

FIG. 2 is a schematic view showing the elements of a single layer withina biofilm structure.

FIG. 3 is a schematic view of a magnified section of a single biofilmlayer.

FIG. 4 is a diagram of a Robbins-type flow cell to measure biofilmdynamics under various flow conditions and components that may be addedto the flowing fluid.

DETAILED DESCRIPTION OF THE INVENTION

P. aeruginosa, which is a gram-negative rod, is one of many organismsfound in slime residues associated with a wide variety of industrial,commercial and processing operations such as sewerage discharges,re-circulating water systems (cooling tower, air conditioning systemsetc.), water condensate collections, paper pulping operations and, ingeneral, any water bearing, handling, processing, collection etc.systems. Just as biofilms are ubiquitous in water handling systems, itis not surprising that P. aeruginosa is also found in association withthese biofilms. In many cases, P. aeruginosa is the major microbialcomponent.

In addition to its importance in industrial processes, P. aeruginosa andits associated biofilm structure has far-reaching medical implications,being the basis of many pathological conditions. P. aeruginosa is anopportunistic bacterium that is associated with a wide variety ofinfections. It has the ability to grow at temperatures higher than manyother bacteria and it is readily transferred from an environmentalsetting to become host-dependent. Translocation, both within a specificmedium and to other media, is facilitated with its single polar flagellagiving it a velocity of 56 μm/sec mobility rate.

P. aeruginosa has nutritional versatility in being able to use a widevariety of substrates, fast growth rate, motility, temperatureresiliency and short incubation periods all of which contribute to itpredominance in natural microflora communities as well as being thecause of nosocomial (hospital acquired) infections.

Infections caused by P. aeruginosa begin usually with bacterialattachment to and colonization of mucosal and cutaneous tissues. Theinfection can proceed via extension to surrounding structures orinfection may lead to bloodstream invasion, dissemination and sepsissyndrome.

Eye Infections. P. aeruginosa colonization in the eye leads to bacterialkeratitis or corneal ulcer and endophthalmitis. Keratitis results fromminor corneal injury, by which, the epithelial surface of the cornea isdisrupted and allows bacterial access to the stroma. Contact lens use,particularly extended wear soft lenses, may exacerbate corneal ulcers.The lens itself or even the lens solution may introduce P. aeruginosainto the eye, while minor lens induced damage to the eye provides theopportunity of infection. Patients exposed to intensive careenvironment, have serious burns or have undergone ocular irradiation areespecially susceptible to P. aeruginosa infections.

Endophthalmitis is a serious intra-ocular infection followingperforation of the cornea, intra-ocular surgery or hematogenous spreadof a previous P. aeruginosa infection.

Respiratory Infections. Alginate producing strains of P. aeruginosainfect the lower respiratory tract of patients with cystic fibrosisleading to acute and the chronic progression of the pathologicalcondition. Primary pneumonia often presents bilateral bronchopneumoniawith nodular infiltrates. Accompanying such infections are pleuraleffusions along with pathological progression leading to alveolarnecrosis, focal hemorrhages and micro-abscesses.

Mucoid strains P. aeruginosa typically infect the lower respiratorytract of individuals with cystic fibrosis. Airway obstruction typicallybegins with bronchial infection and mucus production followed bycolonization of P. aeruginosa in the lower respiratory tract. Thecolonization of P. aeruginosa accelerates disease pathology resulting inincreased mucus production, airway obstruction, bronchiectasis andfibrosis in the lungs. These conditions eventually lead to pulmonarydisease leading to hypertension and hypoxemia.

Ear Infections. P. aeruginosa is a common bacterium residing in the earcanal and is the primary pathogen causing external otitis. A P.aeruginosa infection in the ear canal may present a painful or itchyear, purulent discharge in addition to the canal appearing edematouswith detritus. P. aeruginosa is almost exclusively associated withmalignant external otitis, an invasive condition, associated withdiabetics, in which the infection spreads to surrounding soft tissue,cartilage and bone.

Urinary Tract Infections. P. aeruginosa is the most common causativeagent in complicated and nosocomial urinary tract infections.Opportunities for infection occur during catheterization, surgery,obstruction and bloodborne transfer of P. aeruginosa to the urinarytract. As with other types of P. aeruginosa infections, urinaryinfections tend to be persistent, reoccurring, resistant to antibioticsand chromic in nature.

Skin and Soft Tissue Infections. P. aeruginosa can cause opportunisticinfections in skin and soft tissue in locations where the integrity ofthe tissue is broken by trauma, burn injury, dermatitis and ulcersresulting from peripheral vascular disease. In the case of burn wounds,P. aeruginosa's ability to infect is greatly enhanced due to thebreakdown of the skin, antibiotic selection and burn-related immunedefects.

More specifically, dressings for these types of wounds, as well aswounds in general where an infection can develop, the dressing canincorporate the appropriate enzymes that would degrade initial biofilmformation on these dressings. Such systems are closed systems or mostlyso, and consequently, the enzymes may or may not have moieties attachedto them as a means of retaining them to the would dressing. Further, anadjunct to the embodiment for this application there may also beassociated with it suitable antimicrobial/antibiotic agents.

Endocarditis. P. aeruginosa has been shown to have a high affinity tocardiac tissue including heart valve tissue.

Alginate Biofilms of P. aeruginosa. At the root of P. aeruginosa initialcolonization, as well as its proliferative growth rate, is theproduction of a mucoid exopolysaccharide layer comprised of alginate.This exopolysaccharide layer, along with lipopolysaccharide, protectsthe organism from direct antibody and complement mediated bactericidalmechanisms and from opsonophagocytosis. This protective biofilm allowsP. aeruginosa to expand, grow and to exist in harsh environments thatmay exist outside the alginate biofilm. It is not surprising that thealginate biofilm is considered as an important virulence factor.

The alginate biofilm or “slime matrix” consists of a secretedexopolysaccharide that serves as the backbone structure of the biofilm.Alginate is a polysaccharide copolymer of β-D-mannuronic acid andα-L-guluronic acid linked together by 1-4 linkages. The immediateprecursor to the biosynthetic polymerization is guanosine5′-diphosphate-mannuronic acid, which is converted to mannuronan.Post-polymerization of the mannuronan by acetylation at O-2 and O-3 andepimerization, principally at C-5, of some of the monomeric units toproduce gulonate, results in varying degrees of acetylation and gulonateresidues. Both the degree of acetylation and the percentage ofmannuronic residues that have been converted to gulonate residuesgreatly affect the properties of the biofilm. For example, polymers richin gulonate residues and in the presence of calcium, tend to be morerigid and stiff than polymers with low levels of gulonate monomericunits.

Construction of Anchor-Enzyme Complexes.

The Anchor Enzyme Complex can be constructed using chemical synthetictechniques. Additionally, the Anchor-Enzyme Complex, if the anchor is apolypeptide or protein, such as protein binding domains, lectins,selecting, heparin binding domains etc., can be constructed usingrecombinant genetic engineering techniques.

Types of Anchors.

Elastase binding domain for alginate

-   -   1. Carbohydrate and polysaccharide binding domains    -   2. Lectins    -   3. Selectins    -   4. Heparin binding domains    -   5. Additional anchors listed in U.S. Pat. No. 5,871,714, for        example at column 8 lines 18-67, column 9 lines 1-5.        Types of enzymes

1. Generally, enzymes in the class EC 4.2.2._, which are polysaccharidelyases: EC 3.1.2 Glycoside Hydrolases, Galactoaminidases,Galactosidases, Glucosaminidases, Glucosidases, Mannosidases EC 3.1.2.18Neuraminidase EC 3.2._(—) Dextranase, Mutanase, Mucinase, Amylase,Fructanase, Galactosidase, Muramidase, Levanase, Neuraminidase EC3.2.1.20 α-Glucosidases EC 3.2.1.21 β-Glucosidase EC 3.2.1.22α-Glucosidase EC 3.2.1.25 β-D-Mannosidase EC 3.2.1.30Acetylglucosaminidase EC 3.2.1.35 Hyaluronoglucosaminidase EC 3.2.1.51α-L-Fucosidase EC 4.2.2.1 Hyaluronate Lyase EC 4.2.2.2 Pectate Lyase EC4.2.2.3 Alginate Lyase [Poly(β-D-Mannuronate) Lyase] EC 4.2.2.4Chondroitin ABC Lyase EC 4.2.2.5 Chondroitin AC Lyase EC 4.2.2.6Oligogalacturonide Lyase EC 4.2.2.7 Heparin Lyase EC 4.2.2.8 HeparanLyase [Heparitin-Sulfate Lyase] EC 4.2.2.9 Exopolygalacturonate Lyase EC4.2.2.10 Pectin Lyase EC 4.2.2.11 Poly (α-L-Guluronate) Lyase EC4.2.2.12 Xanthan Lyase EC 4.2.2.13 Exo-(1,4)-α-D-Glucan Lyasefor degrading the polysaccharide backbone structure of biofilms.

2. Enzymes for removing debris embedded within the biofilm structure.These include many EC sub-classes with the general class of hydrolyticand digestive enzymes. In descriptive terms, they include enzymes thatfacilitate the breaking of chemical bonds and include the following:

-   -   Esterases—cleavage of ester bonds;    -   Glycolytic—cleavage of bonds found in oligo- and polysaccharides    -   Peptidases—cleavage of peptide bonds where the substrate is a        protein or polypeptide;    -   Carbon-nitrogen cleavage—where the substrate is not a protein or        polypeptide;    -   Acid anhydride cleaving enzymes;    -   Carbon-carbon bond cleavage;    -   Halide bond cleavage;    -   Phosphorus-nitrogen bond cleavage;    -   Sulfur-nitrogen bond cleavage; and    -   Carbon-phosphorus bond cleavage.

Typical examples include the following enzymes: EC 3.4._(—)Endopeptidases; Peptide Hydrolases EC 3.4.11 Aminopeptidases EC 3.4.11.5Propyl Aminopeptidases EC 3.4.14 Glycylpropyl Dipeptidases; DipeptidylPeptidase EC 3.4.21 Serine Endopeptidases EC 3.4.21.1 Chymotrypsin EC3.4.21.4 Trypsin EC 3.5._(—) Amidohydrolases EC 3.5.1.25N-Acetylglucosamine-6-phosphate Deacetylase EC 4.1.3 Oxo-Acid Lyases EC4.1.3.3 N-Acetylmuraminate Lyases EC 5.1.3_(—) Carbohydrate EpimerasesEC 5.3.1.10 Glucosamine-6-phosphate IsomerasesTypes of Bactericidal Agents

1. Enzymatic

A. Generation of Active Oxygen. Any member from the class ofoxido-reductases, EC 1._that generate active oxygen;

-   -   Monosasccharide oxidases, Peroxidases, Lactoperoxidases,        Salivary peroxidases, Myeloperoxidases, Phenol oxidase,        Cytochrome oxidase, Dioxygenases,Monooxygenases

B. Bacterial cell lytic enzymes

-   -   Lysozyme, Lactoferrin

2. Non-Enzymatic

A. Antimicrobial e.g., chlorhexidine, amine fluoride compounds, fluorideions, hypochlorite, quaterinary ammonium compounds e.g. cetylpyridiniumchloride, hydrogen peroxide, monochloramine, providone iodine, anyrecognized sanitizing agent or oxidative agent and biocides.

B. Antibiotics. Including, but not limited to the following classes andmembers within a class:

Aminoglycosides

-   -   Gentamicin, Tobramycin, Netilmicin, Amikacin, Kanamycin,        Streptomycin, Neomycin

Ouinolones/Fluoroguinolones

-   -   Nalidixic Acid, Cinoxacin, Norfloxacin, Ciprofloxacin,        Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin, Levofloxacin

Antipseudomonal

-   -   Carbenicillin, Carbenicillin Indanyl, Ticarcillin, Azlocillin,    -   Mezlocillin, Piperacillin

Cephalosporins

First Generation

-   -   Cephalothin, Cephaprin, Cephalexin, Cephradine, Cefadroxil,        Cefazolin

Second Generation

-   -   Cefamandole, Cefoxitin, Cefaclor, Cefuroxime, Cefotetan,        Ceforanide, Cefuroxine Axetil, Cefonicid

Third Generation

-   -   Cefotaxime, Moxalactam, Ceftizoxime, Ceftriaxone, Cefoperazone,    -   Cftazidime

Other Cephalosporins

-   -   Cephaloridine, Cefsulodin

Other β-Lactam Antibiotics

-   -   Imipenem, Aztreonam

β-Lactamase Inhibitors

-   -   Clavulanic Acid, Augmentin, Sulbactam

Sulfonamides

-   -   Sulfanilamide, Sulfamethoxazole, Sulfacetamide, Sulfadiazine,        Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide,        p-Aminobenzoic Acid, Trimethoprim-Sulfamethoxazole

Urinary Tract Antiseptics

-   -   Methenamine, Nitrofurantoin, Phenazopyridine and other        napthpyridines

Penicillins

-   -   Penicillin G and Penicillin V

Penicillinase Resistant

-   -   Methicillin, Nafcillin, Oxacillin, Cloxacillin, Dicloxacillin

Penicillins for Gram-Negative/Amino Penicillins

-   -   Ampicillin (Polymycin), Amoxicillin, Cyclacillin, Bacampicillin

Tetracyclines

-   -   Tetracycline, Chlortetracycline, Demeclocycline, Methacycline,        Doxycycline, Minocycline

Other Antibiotics

-   -   Chloramphenicol (Chlormycetin), Erythromycin, Lincomycin,        Clindamycin, Spectinomycin, Polymyxin B (Colistin), Vancomycin,        Bacitracin

Tuberculosis Drugs

-   -   Isoniazid, Rifampin, Ethambutol, Pyrazinamide, Ethinoamide,        Aminosalicylic Acid, Cycloserine

Anti-Fungal Agents

-   -   Amphotericin B. Cyclosporine, Flucytosine

Imidazoles and Triazoles

-   -   Ketoconazole, Miconazaole, Itraconazole, Fluconazole,        Griseofulvin

Topical Anti Fungal Agents

-   -   Clotrimazole, Econazole, Miconazole, Terconazole, Butoconazole,        Oxiconazole, Sulconazole, Ciclopirox Olamine, Haloprogin,        Tolnaftate, Naftifine, Polyene, Amphotericin B, Natamycin

EXAMPLE

Since P. aeruginosa is a ubiquitous bacterial strain, found not only inthe environment and in industrial settings where fouling occurs, butalso in many disease conditions, it will serve as an example toillustrate the principles of the invention. Further, while there aremany disease conditions for which P. aeruginosa is the cause, ocularinfections will exemplify the implementation of the invention. Thechoice of P. aeruginosa as the biofilm-producing bacteria and pathogenand ocular infection as a consequence of the biofilm is not meant topreclude or limit the scope of this invention. The principles outlinedin this example readily apply to all biofilms, whether produced bybacteria or other organisms, all biofilms that are generated byorganisms and the embodiments, taken and implemented either individuallyor collectively.

P. aeruginosa is an opportunistic bacterial species, which oncecolonized at a site such as ocular tissue, produces a biofilm with apolysaccharide-based alginate polymer. This exopolysaccharide orglycocalyx matrix is the confine in which the bacterial species can growand proliferate. This biofilm matrix can also serve as a medium forother, pathogenic bacteria, fungi and viruses. It is of therapeuticbenefit, therefore, to remove the biofilm structure and eliminate allbacteria at the site, not only P. aeruginosa.

Alginate lyase, the expression product from the algL gene, can beobtained from various bacterial sources e.g. Azotobacter vinelandii,Pseudomonas syringe, Pseudomonas aeruginosa etc., producing an enzymeAlgL, which degrades alginate. Other genes, e.g. alxM, also provide awide variety of alginate lyase and polysaccharide depolymerase enzymeswith degrade alginate by various mechanisms.

Endogenous lectins, heparin binding domains and various receptors fromanimals and plants have receptors that bind to alginate. Thesereceptors, when located on host cell surfaces, allow the evolvingalginate biofilm to be retained by the infected tissue. Elastase(Leukocyte Elastase, EC 3.4.21.37 and Pancreatic Elastase, EC3.4.21.36), which is a digestive enzyme, also has a domain that binds toalginate. Such binding capability, along with the degradative ability ofthe catalytic site in elastase, has been implicated in tissuedegradation associated with alginate biofilm infections such as cysticfibrosis. In addition, other serine proteases also have alginate bindingdomains.

In one aspect of the invention, a fusion protein is created, usingstandard genetic engineering techniques. One of the traits or elementsof the fusion protein is the ability to degrade alginate and a secondproperty being a binding capability of the newly-created fusion protein,derived from, for example, the binding domain of elastase. Thebi-functional protein fulfills the criteria set out in the invention inthat the binding domain derived from elastase serves as the anchor andthe alginate lyase portion of the fusion protein serves as thedegradative enzyme for the biofilm.

This embodiment can be used to degrade alginate-based biofilms inindustrial processes where fouling occurs, or implanted medical devices,including catheters and cannulae. This embodiment can also be used for awide variety of infections such as: ophthalmic applications (infections,implants, contact lenses, surgical manipulations etc.), respiratoryinfections, including pneumonia and cystic fibrosis, ear infections,urinary tract infections, skin and soft tissue infections, infectionsthat occur in burn victims, endocarditis, vaginal infections,gastrointestinal tract infections where biofilms, either impair functionor cause infections and in disease conditions, such as cystic fibrosis.

It is within the scope of this invention that the principles outlinedhere also apply to all biofilms in all circumstances in which theyoccur.

Assay Procedure for Synthesized Anchor Enzyme Complexes

Preparation of Bacterial Biofilms. There are many procedures to preparebacterial biofilms. Herein is one of those procedures.

The appropriate bacterial strain, or mixed strains if more than onestrain is used, is incubated in tryptic soy broth for 18 to 24 hours at37° C. After the incubation period, the cells are washed three timeswith isotonic saline and re-suspended in isotonic saline to a density of106 CFU/ml. The re-suspended cells are incubated a second time withTeflon squares (1×1 cm) with a thickness of 0.3 cm for six to seven daysat 37° C. The recovered cells in the saline incubation medium areplanktonic bacteria, while those associated with the Teflon squares andthe biofilm are sessile cells.

The biofilm-associated sessile cells are then treated with appropriateanchor-enzyme complexes that degrade the generated biofilm at variousconcentrations with or without bactericidal agents in either acompletely closed system or an open system (flow-through chamber orcell). The bactericidal agent can be either an anchor enzyme system thatgenerates active oxygen or a non-enzymatic, chemical that is arecognized antimicrobial agent, biocide or antibiotic.

Analysis of a Completely Closed System. The Teflon squares with theassociated biofilm are transferred to isotonic saline medium containinga given concentration of anchor-enzyme complex that degrades thebiofilm. At intervals of 3, 6, 12, 24 and 48 hours, the individualTeflon squares are washed three times with isotonic saline and finallyadded to fresh isotonic saline which is vigorously shaken or sonicatedfor tow minutes. The suspended mixture is diluted and counted for celldensity and expressed as number of CFU/ml.

The same counting procedure can be used for the incubation medium.

Bactericidal agents are also incorporated into the experimental design,which also uses the same cell counting procedure.

Estimating Biofilm Size. At the end of any of the incubation steps, thebiofilm can be recovered, dehydrated and weighed to obtain total biomassof the biofilm. Alternatively, the amount of alginate backbone can bedetermined where the biofilm contains Pseudomonas sp.

Extraction of Polysaccharide Backbone. After the second incubation anddisruption of the biofilm, the bacterial cells are removed from thedispersion. With an increasing concentration of an ethanol/solinggradient, the alginate is precipitated, collected and washed three timeswith 95% ethanol. The precipitate is desiccated after which the quantitycan be determined gravimetrically or by any number of chemical,enzymatic or combination of chemical and enzymatic methods. The mostwidely used method is the chemical method of which there are threetypes: uronic acid assay, orcinol-FeCl3 and decarboxylation and CO2measurement.

Analysis in an Open System (Complete or Partial). The most widely useddynamic flow system that can be regulated from a completely closed to acompletely open system is the Robbins Device or the Modified RobbinsDevice. The Modified Robbins Device allows the assessment of biofilms inwhich the fluid flow and growth rates of the biofilm can be regulatedindependently and simultaneously. A Robbins-type flow cell can be acompletely closed system that possesses flow dynamics for assessingefficacy of anchor-enzyme complexes.

1. A composition for degrading and or removing biofilms and the sessilecells associated therein comprising: an enzyme an anchor moleculecoupled to an enzyme to form an enzyme-anchor complex, the anchor beingcapable of attaching to a surface proximal to a cell colony, the anchorbeing selected from a group consisting of materials that bind to thecellular colony or its components or other bioadhesive molecules;wherein the attachment to the substrate permits prolonged retention timeof the enzyme-anchor complex where the cellular colony and biofilm arepresent.
 2. A composition as claimed in claim 1 wherein the enzyme isselected for its ability to degrade a living cellular colonizing matrix.3. A composition as claimed in claim 1 wherein the anchor enzyme complexis a fusion protein.
 4. A composition as claimed in claim 1 wherein thebiofilm is associated with infections selected from the following:ocular, contact lenses, cystic fibrosis, an implanted device, dermalinfections, oral plaque; industrial equipment and water handlingsystems.
 5. A two component composition comprising an anchor enzymecomplex to degrade biofilm structures and a second anchor enzymecomponent having the capability to act directly upon the bacteria for abactericidal effect.
 6. A composition as claimed in claim 5 wherein theanchor enzyme complex contains alginate lyase to degrade the biofilm. 7.A composition as claimed in claim 5 wherein the anchor enzyme complexcontains an alginate binding domain.
 8. A composition as claimed inclaim 7 wherein the alginate binding domain is derived from elastase. 9.A composition as claimed in claim 5 wherein the anchor enzyme complex isa fusion protein.
 10. A composition as claimed in claim 5 wherein theanchor enzyme complex contains lysozyme to lyse bacteria within thebiofilm.
 11. A composition as claimed in claim 5 wherein the anchorenzyme complex contains lactoferrin.
 12. A composition as claimed inclaim 5 wherein one or more anchor enzyme complexes containoxido-reductase enzymes that generate active oxygen for the purpose ofkilling bacteria within the biofilm.
 13. A composition as claimed inclaim 5 wherein one or more anchor enzyme complexes contain hexoseoxidase for the purpose of generating active oxygen.
 14. A compositionas claimed in claim 5 wherein one or more anchor enzyme complexescontain lactoperoxidase for the purpose of generating active oxygen. 15.A composition as claimed in claim 5 wherein one or more anchor enzymecomplexes contain myeloperoxidase for the purpose of generating activeoxygen.
 16. An ophthalmic composition for treating contact lenses whileeither retained within or removed from the eye consisting of an enzymeanchor complex as claimed in claim
 2. 17. A composition as claimed inclaim 16 wherein the anchor enzyme complex is a fusion protein whoseanchor part is an alginate binding domain and whose catalytic part isalginate lyase.
 18. An ophthalmic composition for treating ocularrelated infections comprising an anchor enzyme complex to degrade thebiofilm associated with the infection and a bactericidal agent to killindividual bacteria that are released from the biofilm structure as itis being degraded.
 19. A composition as claimed in claim 18 wherein thebactericidal agent is selected from the group consisting of:Aminoglycoside antibiotic; a Quinolone or Fluoroquinolone antibiotic, aCephalosporin antibiotic, a Penicillin antibiotic, Tobramycin; isCiprofloxacin, Ofloxacin, Aztreonam, Vancomycin, Streptomycin, isNeomycin, and Gentamicin.
 20. A composition as claimed in claim 19wherein the bactericidal agent has an anchor.
 21. A composition asclaimed in claim 20 wherein the anchor is selected from a polysaccharidebinding domain and a cellulose binding domain.
 22. A composition fordegrading or removing biofilm that is contained in a closed system andthe sessile cells associated therein, the composition comprising one ormore enzymes selected for their ability to degrade the biofilm.
 23. Acomposition as claimed in claim 22 wherein the enzyme is an alginatedegrading enzyme such as alginate lyase and an antimicrobial/antibiotic.24. A composition as claimed in claim 23 wherein theantimicrobial/antibiotic has a moiety connected to it so that theantimicrobial/antibiotic agent can be retained at a specific locationwithin a closed system. 5f. A composition as claimed in claim 22 whereinthe enzyme is an alginate degrading enzyme such as alginate lyase andone or more enzymes that have one or more moieties connected to them sothat the anchor-enzyme can be retained at a specific location within aclosed system.